Influenza vaccine composition

ABSTRACT

An inactivated influenza virus preparation is described which comprises a haemagglutinin antigen stabilised in the absence of thiomersal, or at low levels of thiomersal, wherein the haemagglutinin is detectable by a SRD assay. The influenza virus preparation may comprise a micelle modifying excipient, for example α-tocopherol or a derivative thereof in a sufficient amount to stabilise the haemagglutinin.

[0001] This invention relates to novel influenza virus antigenpreparations, methods for preparing them and their use in prophylaxis ortherapy. In particular the invention relates to inactivated influenzavaccines which are disrupted rather than whole virus vaccines and whichare stable in the absence of organomercurial preservatives. Moreover,the vaccines contain haemagglutinin which is stable according tostandard tests. The vaccines can be administered by any route suitablefor such vaccines, such as intramuscularly, subcutaneously,intradermally or mucosally e.g. intranasally.

[0002] Influenza virus is one of the most ubiquitous viruses present inthe world, affecting both humans and livestock. The economic impact ofinfluenza is significant.

[0003] The influenza virus is an RNA enveloped virus with a particlesize of about 125 nm in diameter. It consists basically of an internalnucleocapsid or core of ribonucleic acid (RNA) associated withnucleoprotein, surrounded by a viral envelope with a lipid bilayerstructure and external glycoproteins. The inner layer of the viralenvelope is composed predominantly of matrix proteins and the outerlayer mostly of host-derived lipid material. The surface glycoproteinsneuraminidase (NA) and haemagglutinin (HA) appear as spikes, 10 to 12 nmlong, at the surface of the particles. It is these surface proteins,particularly the haemagglutinin, that determine the antigenicspecificity of the influenza subtypes.

[0004] Currently available influenza vaccines are either inactivated orlive attenuated influenza vaccine. Inactivated flu vaccines are composedof three possible forms of antigen preparation: inactivated whole virus,sub-virions where purified virus particles are disrupted with detergentsor other reagents to solubilise the lipid envelope (so-called “split”vaccine) or purified HA and NA (subunit vaccine). These inactivatedvaccines are given intramuscularly (i.m.) or intranasaly (i.n.). Thereis no commercially available live attenuated vaccine.

[0005] Influenza vaccines, of all kinds, are usually trivalent vaccines.They generally contain antigens derived from two influenza A virusstrains and one influenza B strain. A standard 0.5 ml injectable dose inmost cases contains 15 μg of haemagglutinin antigen component from eachstrain, as measured by single radial immunodiffusion (SRD) (J. M. Woodet al.: An improved single radial immunodiffusion technique for theassay of influenza haemagglutinin antigen: adaptation for potencydetermination of inactivated whole virus and subunit vaccines. J. Biol.Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborativestudy of single radial diffusion and immunoelectrophoresis techniquesfor the assay of haemagglutinin antigen of influenza virus. J. Biol.Stand. 9 (1981) 317-330).

[0006] The influenza virus strains to be incorporated into influenzavaccine each season are determined by the World Health Organisation incollaboration with national health authorities and vaccinemanufacturers.

[0007] Typical influenza epidemics cause increases in incidence ofpneumonia and lower respiratory disease as witnessed by increased ratesof hospitalisation or mortality. The elderly or those with underlyingchronic diseases are most likely to experience such complications, butyoung infants also may suffer severe disease. These groups in particulartherefore need to be protected.

[0008] Current efforts to control the morbidity and mortality associatedwith yearly epidemics of influenza are based on the use ofintramuscularly administered inactivated influenza vaccines. Theefficacy of such vaccines in preventing respiratory disease andinfluenza complications ranges from 75% in healthy adults to less than50% in the elderly.

[0009] Standards are applied internationally to measure the efficacy ofinfluenza vaccines. The European Union official criteria for aneffective vaccine against influenza are set out in the table below.Theoretically, to meet the European Union requirements, an influenzavaccine has to meet only one of the criteria in the table, for allstrains of influenza included in the vaccine. However in practice, atleast two or all three of the criteria will need to be met for allstrains, particularly for a new vaccine such as a new vaccine fordelivery via a different route. Under some circumstances two criteriamay be sufficient. For example, it may be acceptable for two of thethree criteria to be met by all strains while the third criterion is metby some but not all strains (e.g. two out of three strains). Therequirements are different for adult populations (18-60 years) andelderly populations (>60 years). 18-60 years >60 years Seroconversionrate* >40% >30% Conversion factor** >2.5 >2.0 Protection rate*** >70%>60%

[0010] For a novel flu vaccine to be commercially useful it will notonly need to meet those standards, but also in practice it will need tobe at least as efficacious as the currently available injectablevaccines. It will also need to be commercially viable in terms of theamount of antigen and the number of administrations required.

[0011] The current commercially available influenza vaccines are eithersplit or subunit injectable vaccines. These vaccines are prepared bydisrupting the virus particle, generally with an organic solvent or adetergent, and separating or purifying the viral proteins to varyingextents. Split vaccines are prepared by fragmentation of whole influenzavirus, either infectious or inactivated, with solubilizingconcentrations of organic solvents or detergents and subsequent removalof the solubilizing agent and some or most of the viral lipid material.Split vaccines generally contain contaminating matrix protein andnucleoprotein and sometimes lipid, as well as the membrane envelopeproteins. Split vaccines will usually contain most or all of the virusstructural proteins although not necessarily in the same proportions asthey occur in the whole virus. Subunit vaccines on the other handconsist essentially of highly purified viral surface proteins,haemagglutinin and neuraminidase, which are the surface proteinsresponsible for eliciting the desired virus neutralising antibodies uponvaccination.

[0012] Many vaccines which are currently available require apreservative to prevent deterioration. A frequently used preservative isthimerosal which is a mercury-containing compound. Some public concernshave been expressed about the effects of mercury containing compounds.There is no surveillance system in place to detect the effects of low tomoderate doses of organomercurials on the developing nervous system, andspecial studies of children who have received high doses oforganomercurials will take several years to complete. Certaincommentators have stressed that the potential hazards ofthimerosal-containing vaccines should not be overstated (Offit; P.A.JAMA Vol.283;No:16). Nevertheless, it would be advantageous to findalternative methods for the preparation of vaccines to replace the useof thiomerosal in the manufacturing process. There is thus a need todevelop vaccines which are thimerosal-free, in particular vaccines likeinfluenza vaccines which are recommended, at least for certainpopulation groups, on an annual basis.

[0013] It has been standard practice to date to employ a preservativefor commercial inactivated influenza vaccines, during theproduction/purification process and/or in the final vaccine. Thepreservative is required to prevent microorganisms from growing throughthe various stages of purification. For egg-derived influenza vaccines,thiomersal is typically added to the raw allantoic fluid and may also beadded a second time during the processing of the virus. Thus there willbe residual thiomersal present at the end of the process, and this mayadditionally be adjusted to a desirable preservative concentration inthe final vaccine, for example to a concentration of around 100 μg/ml.

[0014] A side-effect of the use of thiomersal as a preservative in fluvaccines is a stabilisation effect. The thiomersal in commercial fluvaccines acts to stabilise the HA component of the vaccine, inparticular but not exclusively HA of B strain influenza. Certain Astrain haemagglutinins e.g. H3 may also require stabilisation.Therefore, although it may be desirable to consider removing thiomersalfrom influenza vaccines, or at least reducing the concentration of thethiomersal in the final vaccine, there is a problem to overcome in that,without thiomersal, the HA will not be sufficiently stable.

[0015] It has been discovered in the present invention that it ispossible to stabilise HA in inactivated influenza preparations usingalternative reagents that do not contain organomercurials. The HAremains stabilised such that it is detectable over time by quantitativestandard methods, in particular SRD, to an greater extent than anon-stabilised antigen preparation produced by the same method butwithout stabilising excipient. The SRD method is performed as describedhereinabove. Importantly, the HA remains stabilised for up to 12 monthswhich is the standard required of a final flu vaccine.

[0016] In a first aspect the present invention provides an inactivatedinfluenza virus preparation comprising a haemagglutinin antigenstabilised in the absence of thiomersal, or at low levels of thiomersal,wherein the haemagglutinin is detectable by a SRD assay.

[0017] Low levels of thiomersal are those levels at which the stabilityof HA derived from influenza B is reduced, such that a stabilisingexcipient is required for stabilised HA. Low levels of thiomersal aregenerally 5 μg/ml or less.

[0018] Generally, stabilised HA refers to HA which is detectable overtime by quantitative standard methods, in particular SRD, to an greaterextent than a non-stabilised antigen preparation produced by the samemethod but without any stabilising excipient. Stabilisation of HApreferably maintains the activity of HA substantially constant over aone year period. Preferably, stabilisation allows the vaccine comprisingHA to provide acceptable protection after a 6 month storage period, morepreferably a one year period.

[0019] Suitably, stabilisation is carried out by a stabilisingexcipient, preferably a micelle modifying excipient. A micelle modifyingexcipient is generally an excipient that may be incorporated into amicelle formed by detergents used in, or suitable for, solubilising themembrane protein HA, such as the detergents Tween 80, Triton X100 anddeoxycholate, individually or in combination.

[0020] Without wishing to be constrained by theory, it is believed thatthe excipients work to stabilise HA by interaction with the lipids,detergents and/or proteins in the final preparation. Mixed micelles ofexcipient with protein and lipid may be formed, such as micelles ofTween and deoxycholate with residual lipids and/or Triton X-100. It isthought that surface proteins are kept solubilised by those complexmicelles. Preferably, protein aggregation is limited by charge repulsionof micelles containing suitable excipients, such as micelles containingnegatively charged detergents.

[0021] Suitable micelle modifying excipients include: positively,negatively or zwitterionic charged amphiphilic molecules such as alkylsulfates, or alkyl-aryl-sulfates; non-ionic amphiphilic molecules suchas alkyl polyglycosides or derivatives thereof, such as Plantacare®(available from Henkel KGaA), or alkyl alcohol poly alkylene ethers orderivatives thereof such as Laureth-9.

[0022] Preferred excipients are α-tocopherol, or derivatives ofα-tocopherol such as α-tocopherol succinate. Other preferred tocopherolderivatives for use in the invention include D-α tocopherol, D-δtocopherol, D-γ tocopherol and DL-α-tocopherol. Preferred derivatives oftocopherols that may be used include acetates, succinates, phosphoricacid esters, formiates, propionates, butyrates, sulfates and gluconates.Alpha-tocopherol succinate is particularly preferred. The α-tocopherolor derivative is present in an amount sufficient to stabilise thehaemagglutinin.

[0023] Other suitable excipients may be identified by methods standardin the art, and tested for example using the SRD method for stabilityanalysis as described herein.

[0024] In a preferred aspect the invention provides an influenza virusantigen preparation comprising at least one stable influenza B strainhaemagglutinin antigen.

[0025] The invention provides in a further aspect a method for preparinga stable haemagglutinin antigen which method comprises purifying theantigen in the presence of a stabilising micelle modifying excipient,preferably α-tocopherol or a derivative thereof such as α-tocopherolsuccinate.

[0026] Further provided by the invention are vaccines comprising theantigen preparations described herein and their use in a method forprophylaxis of influenza infection or disease in a subject which methodcomprises administering to the subject a vaccine according to theinvention.

[0027] The vaccine may be administered by any suitable delivery route,such as intradermal, mucosal e.g. intranasal, oral, intramuscular orsubcutaneous. Other delivery routes are well known in the art.

[0028] Intradermal delivery is preferred. Any suitable device may beused for intradermal delivery, for example short needle devices such asthose described in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521,U.S. Pat. No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No.4,270,537, U.S. Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat.No. 5,417,662. Intradermal vaccines may also be administered by deviceswhich limit the effective penetration length of a needle into the skin,such as those described in WO99/34850 and EP1092444, incorporated hereinby reference, and functional equivalents thereof. Also suitable are jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector or via a needle which pierces the stratum corneumand produces a jet which reaches the dermis. Jet injection devices aredescribed for example in U.S. Pat. No. 5,480,381, U.S. Pat. No.5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat.No. 5,649,912, U.S. Pat. No. 5,569,189, U.S. Pat. No. 5,704,911, U.S.Pat. No. 5,383,851, U.S. Pat. No. 5,893,397, U.S. Pat. No. 5,466,220,U.S. Pat. No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No.5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat. No. 5,520,639, U.S. Pat.No. 4,596,556U.S. Pat. No. 4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat.No. 4,940,460, WO 97/37705 and WO 97/13537. Also suitable are ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis. Additionally, conventional syringes may be used in the classicalmantoux method of intradermal dministration. However, the use ofconventional syringes requires highly skilled operators and thus deviceswhich are capable of accurate delivery without a highly skilled user arepreferred.

[0029] The invention thus provides a method for the prophylaxis ofinfluenza infection or disease in a subject which method comprisesadministering to the subject intradermally an influenza vaccineaccording to the invention.

[0030] The invention also extends to intradermal devices in combinationwith a vaccine according to the present invention, in particular withdevices disclosed in WO99/34850 or EP1092444, for example.

[0031] Also provided is the use of a micelle modifying excipient,preferably α-tocopherol or a derivative thereof as a haemagglutininstablilser in the manufacture of an influenza vaccine.

[0032] The invention applies particularly but not exclusively to thestabilisation of B strain influenza haemagglutinin.

[0033] Preferably the stabilised HA of the present invention is stablefor 6 months, more preferably 12 months.

[0034] Preferably the α-tocopherol is in the form of an ester, morepreferaby the succinate or acetate and most preferably the succinate.

[0035] Preferred concentrations for the α-tocopherol or derivative arebetween 1 μg/ml-10 mg/ml, more preferably between 10 μg/ml-500 μg/ml.

[0036] The vaccine according to the invention generally contains both Aand B strain virus antigens, typically in a trivalent composition of twoA strains and one B strain. However, divalent and monovalent vaccinesare not excluded. Monovalent vaccines may be advantageous in a pandemicsituation, for example, where it is important to get as much vaccineproduced and administered as quickly as possible.

[0037] The non-live flu antigen preparation for use in the invention maybe selected from the group consisting of split virus antigenpreparations, subunit antigens (either recombinantly expressed orprepared from whole virus), inactivated whole virus which may bechemically inactivated with e.g. formaldehyde, β-propiolactone orotherwise inactivated e.g. U.V. or heat inactivated. Preferably theantigen preparation is either a split virus preparation, or a subunitantigen prepared from whole virus, particularly by a splitting processfollowed by purification of the surface antigen. Most preferred aresplit virus preparations.

[0038] Preferably the concentration of haemagglutinin antigen for the oreach strain of the influenza virus preparation is 1-1000 μg per ml, morepreferably 3-300 μg per ml and most preferably about 30 μg per ml, asmeasured by a SRD assay.

[0039] The vaccine according to the invention may further comprise anadjuvant or immunostimulant such as but not limited to detoxified lipidA from any source and non-toxic derivatives of lipid A, saponins andother reagents capable of stimulating a TH1 type response.

[0040] It has long been known that enterobacterial lipopolysaccharide(LPS) is a potent stimulator of the immune system, although its use inadjuvants has been curtailed by its toxic effects. A non-toxicderivative of LPS, monophosphoryl lipid A (MPL), produced by removal ofthe core carbohydrate group and the phosphate from the reducing-endglucosamine, has been described by Ribi et al (1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY,p407-419) and has the following structure:

[0041] A further detoxified version of MPL results from the removal ofthe acyl chain from the 3-position of the disaccharide backbone, and iscalled 3-O-Deacylated monophosphoryl lipid A (3D-MPL). It can bepurified and prepared by the methods taught in GB 2122204B, whichreference also discloses the preparation of diphosphoryl lipid A, and3-O-deacylated variants thereof.

[0042] A preferred form of 3D-MPL is in the form of an emulsion having asmall particle size less than 0.2 μm in diameter, and its method ofmanufacture is disclosed in WO 94/21292. Aqueous formulations comprisingmonophosphoryl lipid A and a surfactant have been described inWO9843670A2.

[0043] The bacterial lipopolysaccharide derived adjuvants to beformulated in the compositions of the present invention may be purifiedand processed from bacterial sources, or alternatively they may besynthetic. For example, purified monophosphoryl lipid A is described inRibi et al 1986 (supra), and 3-O-Deacylated monophosphoryl ordiphosphoryl lipid A derived from Salmonella sp. is described in GB2220211 and U.S. Pat. No. 4,912,094. Other purified and syntheticlipopolysaccharides have been described (Hilgers et al., 1986,Int.Arch.Allergy.Immunol., 79(4):392-6; Hilgers et al., 1987,Immunology, 60(1):141-6; and EP 0 549 074 B1). A particularly preferredbacterial lipopolysaccharide adjuvant is 3D-MPL.

[0044] Accordingly, the LPS derivatives that may be used in the presentinvention are those immunostimulants that are similar in structure tothat of LPS or MPL or 3D-MPL. In another aspect of the present inventionthe LPS derivatives may be an acylated monosaccharide, which is asub-portion to the above structure of MPL.

[0045] Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. Areview of the biological and pharmacological activities of saponins.Phytomedicine vol 2 pp 363-386). Saponins are steroid or triterpeneglycosides widely distributed in the plant and marine animal kingdoms.Saponins are noted for forming colloidal solutions in water which foamon shaking, and for precipitating cholesterol. When saponins are nearcell membranes they create pore-like structures in the membrane whichcause the membrane to burst. Haemolysis of erythrocytes is an example ofthis phenomenon, which is a property of certain, but not all, saponins.

[0046] Saponins are known as adjuvants in vaccines for systemicadministration. The adjuvant and haemolytic activity of individualsaponins has been extensively studied in the art (Lacaille-Dubois andWagner, supra). For example, Quil A (derived from the bark of the SouthAmerican tree Quillaja Saponaria Molina), and fractions thereof, aredescribed in U.S. Pat. No. 5,057,540 and “Saponins as vaccineadjuvants”, Kensil, C. R., Crit Rev Ther Drug Carrier Syst, 1996, 12(1-2):1-55; and EP 0 362 279 B1. Particulate structures, termed ImmuneStimulating Complexes (ISCOMS), comprising fractions of Quil A arehaemolytic and have been used in the manufacture of vaccines (Morein,B., EP 0 109 942 B1; WO 96/11711; WO 96/33739). The haemolytic saponinsQS21 and QS17 (HPLC purified fractions of Quil A) have been described aspotent systemic adjuvants, and the method of their production isdisclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B 1. Othersaponins which have been used in systemic vaccination studies includethose derived from other plant species such as Gypsophila and Saponaria(Bomford et al., Vaccine, 10(9):572-577, 1992).

[0047] An enhanced system involves the combination of a non-toxic lipidA derivative and a saponin derivative particularly the combination ofQS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol as disclosed inWO 96/33739.

[0048] A particularly potent adjuvant formulation involving QS21 and3D-MPL in an oil in water emulsion is described in WO 95/17210 and is apreferred formulation.

[0049] Accordingly in one embodiment of the present invention there isprovided a vaccine comprising an influenza antigen preparation of thepresent invention adjuvanted with detoxified lipid A or a non-toxicderivative of lipid A, more preferably adjuvanted with a monophosphoryllipid A or derivative thereof.

[0050] Preferably the vaccine additionally comprises a saponin, morepreferably QS21.

[0051] Preferably the formulation additionally comprises an oil in wateremulsion. The present invention also provides a method for producing avaccine formulation comprising mixing an antigen preparation of thepresent invention together with a pharmaceutically acceptable excipient,such as 3D-MPL.

[0052] The vaccines according to the invention may further comprise atleast one surfactant which may be in particular a non-ionic surfactant.Suitable non-ionic surfactant are selected from the group consisting ofthe octyl- or nonylphenoxy polyoxyethanols (for example the commerciallyavailable Triton™ series), polyoxyethylene sorbitan esters (Tween™series) and polyoxyethylene ethers or esters of general formula (I):

HO(CH₂CH₂O)_(n)-A-R  (I)

[0053] wherein n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl orphenyl C₁₋₅₀ alkyl; and combinations of two or more of these.

[0054] Preferred surfactants falling within formula (I) are molecules inwhich n is 4-24, more preferably 6-12, and most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₋₂alkyl.

[0055] Octylphenoxy polyoxyethanols and polyoxyethylene sorbitan estersare described in “Surfactant systems” Eds: Attwood and Florence (1983,Chapman and Hall). Octylphenoxy polyoxyethanols (the octoxynols),including t-octylphenoxypolyethoxyethanol (Triton X-100™) are alsodescribed in Merck Index Entry 6858 (Page 1162, 12^(th) Edition, Merck &Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Thepolyoxyethylene sorbitan esters, including polyoxyethylene sorbitanmonooleate (Tween 80™) are described in Merck Index Entry 7742 (Page1308, 12^(th) Edition, Merck & Co. Inc., Whitehouse Station, N.J., USA;ISBN 0911910-12-3). Both may be manufactured using methods describedtherein, or purchased from commercial sources such as Sigma Inc.

[0056] Particularly preferred non-ionic surfactants include Triton X-45,t-octylphenoxy polyethoxyethanol (Triton X-100), Triton X-102, TritonX-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, TritonN-101, Triton N-128, Breij 35, polyoxyethylene-9-lauryl ether (laureth9) and polyoxyethylene-9-stearyl ether (steareth 9). Triton X-100 andlaureth 9 are particularly preferred. Also particularly preferred is thepolyoxyethylene sorbitan ester, polyoxyethylene sorbitan monooleate(Tween 80™).

[0057] Further suitable polyoxyethylene ethers of general formula (I)are selected from the following group: polyoxyethylene-8-stearyl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

[0058] Alternative terms or narnes for polyoxyethylene lauryl ether aredisclosed in the CAS registry. The CAS registry number ofpolyoxyethylene-9 lauryl ether is: 9002-92-0. Polyoxyethylene etherssuch as polyoxyethylene lauryl ether are described in the Merck index(12^(th) ed: entry 7717, Merck & Co. Inc., Whitehouse Station, N.J.,USA; ISBN 0911910-12-3). Laureth 9 is formed by reacting ethylene oxidewith dodecyl alcohol, and has an average of nine ethylene oxide units.

[0059] Two or more non-ionic surfactants from the different groups ofsurfactants described may be present in the vaccine formulationdescribed herein. In particular, a combination of a polyoxyethylenesorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80™)and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton) X-100™is preferred. Another particularly preferred combination of non-ionicsurfactants comprises laureth 9 plus a polyoxyethylene sorbitan ester oran octoxynol or both.

[0060] Non-ionic surfactants such as those discussed above havepreferred concentrations in the final vaccine composition as follows:polyoxyethylene sorbitan esters such as Tween 80™: 0.01 to 1%, mostpreferably about 0.1% (w/v); octyl- or nonylphenoxy polyoxyethanols suchas Triton X-100™ or other detergents in the Triton series: 0.001 to0.1%, most preferably 0.005 to 0.02% (w/v); polyoxyethylene ethers ofgeneral formula (I) such as laureth 9: 0.1 to 20%, preferably 0.1 to 10%and most preferably 0.1 to 1% or about 0.5% (w/v).

[0061] For certain vaccine formulations, other vaccine components may beincluded in the formulation. As such the formulations of the presentinvention may also comprise a bile acid or a derivative thereof, inparticular in the form of a salt. These include derivatives of cholicacid and salts thereof, in particular sodium salts of cholic acid orcholic acid derivatives. Examples of bile acids and derivatives thereofinclude cholic acid, deoxycholic acid, chenodeoxycholic acid,lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid andderivatives such as glyco-, tauro-, amidopropyl-1-propanesulfonic-,amidopropyl-2-hydroxy-1-propanesulfonic derivatives of theaforementioned bile acids, or N,N-bis (3Dgluconoamidopropyl)deoxycholamide. A particularly preferred example is sodium deoxycholate(NaDOC) which may be present in the final vaccine dose.

[0062] Also provided by the invention are pharmaceutical kits comprisinga vaccine administration device filled with a vaccine according to theinvention. Such administration devices include but are not limited toneedle devices, liquid jet devices, powder devices, and spray devices(for intranasal use).

[0063] The influenza virus antigen preparations according to theinvention may be derived from the conventional embryonated egg method,or they may be derived from any of the new generation methods usingtissue culture to grow the virus or express recombinant influenza virussurface antigens. Suitable cell substrates for growing the virus includefor example dog kidney cells such as MDCK or cells from a clone of MDCK,MDCK-like cells, monkey kidney cells such as AGMK cells including Verocells, suitable pig cell lines, or any other mammalian cell typesuitable for the production of influenza virus for vaccine purposes.Suitable cell substrates also include human cells e.g. MRC-5 cells.Suitable cell substrates are not limited to cell lines; for exampleprimary cells such as chicken embryo fibroblasts are also included.

[0064] The influenza virus antigen preparation may be produced by any ofa number of commercially applicable processes, for example the split fluprocess described in patent no. DD 300 833 and DD 211 444, incorporatedherein by reference. Traditionally split flu was produced using asolvent/detergent treatment, such as tri-n-butyl phosphate, ordiethylether in combination with Tween™ (known as “Tween-ether”splitting) and this process is still used in some production facilities.Other splitting agents now employed include detergents or proteolyticenzymes or bile salts, for example sodium deoxycholate as described inpatent no. DD 155 875, incorporated herein by reference. Detergents thatcan be used as splitting agents include cationic detergents e.g. cetyltrimethyl ammonium bromide (CTAB), other ionic detergents e.g.laurylsulfate, taurodeoxycholate, or non-ionic detergents such as theones described above including Triton X-100 (for example in a processdescribed in Lina et al, 2000, Biologicals 28, 95-103) and Triton N-101,or combinations of any two or more detergents.

[0065] The preparation process for a split vaccine will include a numberof different filtration and/or other separation steps such asultracentrifugation, ultrafiltration, zonal centrifugation andchromatography (e.g. ion exchange) steps in a variety of combinations,and optionally an inactivation step eg with heat, formaldehyde orpropiolactone or U.V. which may be carried out before or aftersplitting. The splitting process may be carried out as a batch,continuous or semi-continuous process.

[0066] Preferred split flu vaccine antigen preparations according to theinvention comprise a residual amount of Tween 80 and/or Triton X-100remaining from the production process, although these may be added ortheir concentrations adjusted after preparation of the split antigen.Preferably both Tween 80 and Triton X-100 are present. The preferredranges for the final concentrations of these non-ionic surfactants inthe vaccine dose are:

[0067] Tween 80: 0.01 to 1%, more preferably about 0.1% (v/v)

[0068] Triton X-100: 0.001 to 0.1 (% w/v), more preferably 0.005 to0.02% (w/v).

[0069] Alternatively the influenza virus antigen preparations accordingto the invention may be derived from a source other than the liveinfluenza virus, for example the haemagglutinin antigen may be producedrecombinantly.

[0070] The invention will now be further described in the following,non-limiting examples.

EXAMPLES Example 1 Preparation of Influenza Virus Antigen PreparationUsing α-Tocopherol Succinate as a Stabiliser for a Preservative-FreeVaccine (Thiomersal-Reduced Vaccine)

[0071] Monovalent split vaccine was prepared according to the followingprocedure.

[0072] Preparation of Virus Inoculum

[0073] On the day of inoculation of embryonated eggs a fresh inoculum isprepared by mixing the working seed lot with a phosphate buffered salinecontaining gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25μg/ml. (virus strain-dependent). The virus inoculum is kept at 2-8° C.

[0074] Inoculation of Embryonated Eggs

[0075] Nine to eleven day old embryonated eggs are used for virusreplication. Shells are decontaminated. The eggs are inoculated with 0.2ml of the virus inoculum. The inoculated eggs are incubated at theappropriate temperature (virus strain-dependent) for 48 to 96 hours. Atthe end of the incubation period, the embryos are killed by cooling andthe eggs are stored for 12-60 hours at 2-8° C.

[0076] Harvest

[0077] The allantoic fluid from the chilled embryonated eggs isharvested. Usually, 8 to 10 ml of crude allantoic fluid is collected peregg.

[0078] Concentration and Purification of Whole Virus From AllantoicFluid

[0079] 1. Clarification

[0080] The harvested allantoic fluid is clarified by moderate speedcentrifugation (range: 4000-14000 g).

[0081] 2. Adsorption step

[0082] To obtain a CaHPO₄ gel in the clarified virus pool, 0.5 mol/LNa₂HPO₄ and 0.5 mol/L CaCl₂ solutions are added to reach a finalconcentration of CaHPO₄ of 1.5 g to 3.5 g CaHPO₄/litre depending on thevirus strain.

[0083] After sedimentation for at last 8 hours, the supernatant isremoved and the sediment containing the influenza virus is resolubilisedby addition of a 0.26 mol/L EDTA-Na₂ solution, dependent on the amountof CaHPO₄ used.

[0084] 3. Filtration

[0085] The resuspended sediment is filtered on a 6 μm filter membrane.

[0086] 4. Sucrose Gradient Centrifugation

[0087] The influenza virus is concentrated by isopycnic centrifugationin a linear sucrose gradient (0.55% (w/v)) containing 100 μg/mlThiomersal. The flow rate is 8-15 litres/hour.

[0088] At the end of the centrifugation, the content of the rotor isrecovered by four different fractions (the sucrose is measured in arefractometer): fraction 1 55-52% sucrose fraction 2 approximately52-38% sucrose fraction 3 38-20% sucrose* fraction 4 20-0% sucrose

[0089] For further vaccine preparation, only fractions 2 and 3 are used.

[0090] Fraction 3 is washed by diafiltration with phosphate buffer inorder to reduce the sucrose content to approximately below 6%. Theinfluenza virus present in this diluted fraction is pelleted to removesoluble contaminants.

[0091] The pellet is resuspended and thoroughly mixed to obtain ahomogeneous suspension. Fraction 2 and the resuspended pellet offraction 3 are pooled and phosphate buffer is added to obtain a volumeof approximately 40 litres. This product is the monovalent whole virusconcentrate.

[0092] 5. Sucrose Gradient Centrifugation with Sodium Deoxycholate

[0093] The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v) and Tocopherol succinate is added for B-strain-viruses up to 0.5mM. The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v) andis strain dependent. The flow rate is 8-15 litres/hour.

[0094] At the end of the centrifugation, the content of the rotor isrecovered by three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

[0095] 6. Sterile Filtration

[0096] The split virus fraction is filtered on filter membranes endingwith a 0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween80 and (for B strain viruses) 0.5 mM Tocopherol succinate is used fordilution. The final volume of the filtered fraction 2 is 5 times theoriginal fraction volume.

[0097] 7. Inactivation

[0098] The filtered monovalent material is incubated at 22±2° C. for atmost 84 hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffer containing 0.025% (w/v). Tween 80 is thenadded in order to reduce the total protein content down to max. 250μg/ml. For B strain viruses, a phosphate buffered saline containing0.025% (w/v) Tween 80 and 0.25 mM Tocopherol succinate is applied fordilution to reduce the total protein content down to 250 μg/ml.Formaldehyde is added to a final concentration of 50 μg/ml and theinactivation takes place at 20° C.±2° C. for at least 72 hours.

[0099] 8. Ultrafiltration

[0100] The inactivated split virus material is concentrated at least 2fold in a ultrafiltration unit, equipped with cellulose acetatemembranes with 20 kDa MWCO. The Material is subsequently washed withphosphate buffer containing 0.025% (w/v) Tween 80 and following withphosphate buffered saline containing 0.01% (w/v) Tween. For B strainvirus a phosphate buffered saline containing 0.01% (w/v) Tween 80 and0.1 mM Tocopherol succinate is used for washing.

[0101] 9. Final Sterile Filtration

[0102] The material after ultrafiltration is filtered on filtermembranes ending with a 0.2 μm membrane. Filter membranes are rinsed andthe material is diluted if necessary such that the protein concentrationdoes not exceed 500 μg/ml with phosphate buffered saline containing0.01% (w/v) Tween 80 and (for B strain viruses) 0.1 mM Tocopherolsuccinate.

[0103] 10. Storage

[0104] The monovalent final bulk is stored at 2-8° C. for a maximum of18 months.

[0105] Stability TABLE 1 Comparison of time dependent HA content (μg/ml)measured by SRD in monovalent final bulks. After 4 weeks 6 month 12month at Strain Stabiliser production at 30° C. at 2-8° C. 2-8° C.B/Yamanashi/166/98 Tocopherylsuccinate 169 139 172 ND (residual mercury3 μg/ml) (82%) (>100%) B/Yamanashi/166/98 Thiomersal 192 160 186 178(108 μg/ml) (83%) (97%) (93%) B/Yamanashi/166/98 None 191 122 175 154(residual mercury 3 μg/ml) (60%) (92%) (81%) B/Johannesburg/5/99Tocopherylsuccinate 166 183 (>100%) 158 179 (residual mercury 4 μg/ml)(95%) (>100%) B/Johannesburg/5/99 Tocopherylsuccinate 167 179 158 178(residual mercury 4 μg/ml) (>100%) (95%) (>100%) B/Johannesburg/5/99Tocopherylsuccinate 144 151 130 145 (residual mercury 3 μg/ml) (>100%)(90%) (>100%) B/Johannesburg/5/99* Thiomersal 159 ND 172 154 (>100%)(97%) B/Johannesburg/5/99** None 169 107 153 ON (63%) (90%)

Example 2 Preparation of Influenza Vaccine Using α-Tocopherol Succinateas a Stabiliser for a Thiomersal-Reduced Vaccine

[0106] Monovalent final bulks of three strains, A/New Caldonia/20/99(H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17 and B/Yamanashi/166/98were produced according to the method described in Example 1.

[0107] Pooling

[0108] The appropriate amount of monovalent final bulks were pooled to afinal HA-concentration of 30 μg/ml for A/New Caldonia/20/99 (H1N1)IVR-116, A/Panama/2007/99 (H₃N₂) Resvir-17, respectively and of 39 μg/mlfor B/Yamanashi/166/98. Tween 80 and Triton X-100 were adjusted to 580μg/ml and 90 μg/ml, respectively. The final volume was adjusted to 3 lwith phosphate buffered saline. The trivalent pool was filtered endingwith 0.8 μm cellulose acetate membrane to obtain the trivalent finalbulk. Trivalent final bulk was filled into syringes at least 0.5 mL ineach. TABLE 2 Comparison of time dependent HA content measured by SRD intrivalent final bulks which was recovered from syringes. 0 2 4 6 Vaccineformul. Strain months months months months Influenza vaccine withoutA/NCal/20/99 33 (32-34) 32 (31-33) 36 (34-38) 31 (30-32) stabilizerA/Pan/2007/99 29 (27-31) 31 (28-34) 34 (32-36) 32 (31-33) B/Yam/166/9836 (34-38) 33 (32-34) 32 (30-34) 31 (29-33) Influenza vaccineA/NCal/20/99 31 (30-32) 32 (31-33) 36 (34-38) 32 (31-33) containingalpha-tocopherol succinate A/Pan/2007/99 33 (30-36) 33 (30-36) 36(35-37) 33 (31-35) B/Yam/166/98 37 (35-39) 36 (34-38) 38 (35-41) 36(33-39)

Example 3 SRD Method Used to Measure Haemagglutinin Content

[0109] Glass plates (12.4-10.0 cm) are coated with an agarose gelcontaining a concentration of anti-influenza HA serum that isrecommended by NIBSC. After the gel has set, 72 sample wells (3 mmØ) arepunched into the agarose. 10 microliters of appropriate dilutions of thereference and the sample are loaded in the wells. The plates areincubated for 24 hours at room temperature (20 to 25° C.) in a moistchamber. After that, the plates are soaked overnight with NaCl-solutionand washed briefly in distilled water. The gel is then pressed anddried. When completely dry, the plates are stained on Coomassie BrillantBlue solution for 10 min and destained twice in a mixture of methanoland acetic acid until clearly defined stained zones become visible.After drying the plates, the diameter of the stained zones surroundingantigen wells is measured in two directions at right angles.Alternatively equipment to measure the surface can be used.Dose-response curves of antigen dilutions against the surface areconstructed and the results are calculated according to standardslope-ratio assay methods (Finney, D. J. (1952). Statistical Methods inBiological Assay. London: Griffin, Quoted in: Wood, μM, et al (1977). J.Biol. Standard. 5, 237-247).

Example 4 Clinical Testing of α-Tocopherol Stabilised Influenza Vaccine(Reduced Thiomersal)

[0110] Syringes obtained as described in Example 2 are used for clinicaltesting

[0111] H₃N₂: A/Panama/2007/99 RESVIR-17

[0112] H1N1: A/New Calcdonia/20/99 (H1N1) IVR-116

[0113] B: B/Yamanashi/166/98 TABLE 3 thio- thio- Adults 18-60 reducedplus years H3N2 H1N1 B H3N2 H1N1 B pre- GMT  47  41 111  55  37  102vacc. Titer <10 [%]  10.3%  13.8%  1.7%  5.3%  12.3%  8.8% Titer ≧40,SPR [%]  60.3%  55.2%  75.9%  70.2%  52.6%  75.4% post- Seroconv. rate[%]  10.3%  13.8%  1.7%  5.3%  12.3%  8.8% vacc. Significant  58.6% 74.1%  58.6%  63.2%  73.7%  52.6% Increase in antibody titer [%]Seroconversions [%]  58.6%  74.1%  58.6%  63.2%  73.7%  52.6% GMT 328525 766 324 359 588 Fold GMT  7.3  13.0  6.9  5.9  9.8  5.9 Titer ≧40,SPR [%] 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

Example 5a Preparation Influenza Virus Antigen Preparation Usingα-Tocopherol Succinate as a Stabiliser for a Thiomersal-Free Vaccine

[0114] Monovalent split vaccine was prepared according to the followingprocedure.

[0115] Preparation of Virus Inoculum

[0116] On the day of inoculation of embryonated eggs a fresh inoculum isprepared by mixing the working seed lot with a phosphate buffered salinecontaining gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25μg/ml. (virus strain-dependent). The virus inoculum is kept at 2-8° C.

[0117] Inoculation of Embryonated Eggs

[0118] Nine to eleven day old embryonated eggs are used for virusreplication. Shells are decontaminated. The eggs are inoculated with 0.2ml of the virus inoculum. 60,000 inoculated eggs are incubated at theappropriate temperature (virus strain-dependent) for 48 to 96 hours. Atthe end of the incubation period, the embryos are killed by cooling andthe eggs are stored for 12-60 hours at 2-8° C.

[0119] Harvest

[0120] The allantoic fluid from the chilled embryonated eggs isharvested. Usually, 8 to 10 ml of crude allantoic fluid is collected peregg.

[0121] Concentration and Purification of Whole Virus From AllantoicFluid

[0122] Clarification

[0123] The harvested allantoic fluid is clarified by moderate speedcentrifugation (range: 4000-14000 g).

[0124] Precipitation Step

[0125] Saturated ammonium sulfate solution is added to the clarifiedvirus pool to reach a final ammonium sulfate concentration of 0.5 mol/L.After sedimentation for at least 1 hour, the precipitate is removed byfiltration on depth filters (typically 0.5 μm)

[0126] Filtration

[0127] The clarified crude whole virus bulk is filtered on filtermembranes ending with a validated sterile membrane (typically 0.2 μm).

[0128] Ultrafiltration

[0129] The sterile filtered crude monovalent whole virus bulk isconcentrated on a cassettes equipped with 1000 kDa MWCO BIOMAX™ membraneat least 6 fold. The concentrated retentate is washed with phosphatebuffered saline at least 1.8 times.

[0130] Sucrose Gradient Centrifugation

[0131] The influenza virus is concentrated by isopycnic centrifugationin a linear sucrose gradient (0.55% (w/v)). The flow rate is 8-15litres/hour.

[0132] At the end of the centrifugation, the content of the rotor isrecovered by four different fractions (the sucrose is measured in arefractometer): fraction 1 55-52% sucrose fraction 2 approximately52-38% sucrose fraction 3 38-20% sucrose* fraction 4 20-0% sucrose

[0133] For further vaccine preparation, either only fractions 2 is usedor fraction 2 together with a further purified fraction 3 are used.

[0134] Fraction 3 is washed by diafiltration with phosphate buffer inorder to reduce the sucrose content to approximately below 6%.Optionally this step may be omitted. The influenza virus present in thisdiluted fraction is pelleted to remove soluble contaminants.

[0135] The pellet is resuspended and thoroughly mixed to obtain ahomogeneous suspension. Fraction 2 and the resuspended pellet offraction 3 are pooled and phosphate buffer is added to obtain a volumeof approximately 40 litres. This product is the monovalent whole virusconcentrate.

[0136] Sucrose Gradient Centrifugation with Sodium Deoxycholate

[0137] The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v) and Tocopherylsuccinate is added for B-strain viruses up to 0.5mM. The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v) andis strain dependent. The flow rate is 8-15 litres/hour.

[0138] At the end of the centrifugation, the content of the rotor isrecovered by three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

[0139] Sterile Filtration

[0140] The split virus fraction is filtered on filter membranes endingwith a 0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween80 and (for B strains) 0.5 mM Tocopherylsuccinate is used for dilution.The final volume of the filtered fraction 2 is 5 times the originalfraction volume.

[0141] Inactivation

[0142] The filtered monovalent material is incubated at 22±2° C. for atmost 84 hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffer containing 0.025% (w/v) Tween 80 is thenadded in order to reduce the total protein content down to max. 450μg/ml. For B-strains a phosphate buffered saline containing 0.025% (w/v)Tween 80 and 0.25 mM Tocopherylsuccinate is applied for dilution toreduce the total protein content down to 450 μg/ml. Formaldehyde isadded to a final concentration of 100 μg/ml and the inactivation takesplace at 20° C. ±2° C. for at least 72 hours.

[0143] Ultrafiltration

[0144] The inactivated split virus material is concentrated at least 2fold in a ultrafiltration unit, equipped with cellulose acetatemembranes with 20 kDa MWCO. The Material is subsequently washed withphosphate buffer containing 0.025% (w/v) Tween 80 and following withphosphate buffered saline containing 0.01% (w/v) Tween. For B-strainviruses a phosphate buffered saline containing 0.01% (w/v) Tween 80 and0.1 mM Tocopherylsuccinate is used for washing.

[0145] Final Sterile Filtration

[0146] The material after ultrafiltration is filtered on filtermembranes ending with a 0.2 μm membrane. Filter membranes are rinsed andthe material is diluted if necessary that the protein concentration doesnot exceed 500 μg/ml with phosphate buffered saline containing 0.01%(w/v) Tween 80 and, specific for B strains, 0.1 mM Tocopherylsuccinate.

[0147] Storage

[0148] The monovalent final bulk is stored at 2-8° C. for a maximum of18 months.

[0149] Stability TABLE 4 Comparison of time dependent HA content (μg/ml)measured by SRD in monovalent final bulks. After 4 weeks 6 month StrainStabiliser production at 30° C. at 2-8° C. B/Johannesburg/5/99Tocopherol 214 196 206 succinate (92%) (96%) B/Johannesburg/5/ None 169107 153 99** (63%) (90%)

Example 5b Preparation of Influenza Virus Antigen Preparation Usingα-Tocopherol Succinate as a Stabiliser for a Thiomersal-Free Vaccine

[0150] A preferred variation of the method described in Example 5a is asfollows:

[0151] Harvesting of the whole virus is followed by the precipitationstep (ammonium sulfate precipitation). This is followed by theclarification step where the fluid is clarified by moderate speedcentrifugation (range 4000-14000 g). Thus the order of the precipitationand clarification steps is reversed compared to Example 5a.

[0152] Sterile filtration, ultrafiltration and ultracentrifugation(sucrose gradient centrifugation) steps follow as for Example 5a.However, there is no need for reprocessing step of the fractionsresulting from the ultracentrifugation step.

[0153] The remaining steps in the process are as described in Example5a.

[0154] Thus, the summarised process in this example is as follows:

[0155] Harvest

[0156] Precipitation (ammonium sulfate)

[0157] Clarification

[0158] Sterile filtration

[0159] Ultrafiltration

[0160] Ultracentrifugation

[0161] Splitting (preferably sodium deoxycholate)

[0162] Sterile filtration

[0163] Inactivation

[0164] Ultrafiltration

[0165] Final sterile filtration

[0166] Another preferred variation of Example 5a involves aprefiltration step before the first sterile filtration. This uses amembrane which does not sterile filter but which enables the removal ofcontaminants e.g. albumin prior to sterile filtration. This can resultin a better yield. A suitable membrane for prefiltration is about 0.8 μmto about 1.8 μm, for example 1.2 μm. The prefiltration step can be usedin the scheme of Example 5a or Example 5b.

Example 6 Preparation of Influenza Vaccine Using α-Tocopherol Succinateas a Stabiliser for a Thiomersal-Free Vaccine

[0167] Monovalent final bulks of three strains, A/New Caldonia/20/99(H1N1) IVR-116, A/Panama/2007/99 (H₃N₂) Resvir-17 and B/Yamanashi/166/98were produced according to the method described in Example 5.

[0168] Pooling

[0169] The appropriate amount of monovalent final bulks were pooled to afinal HA-concentration of 30 μg/ml for A/New Caldonia/20/99 (H1N1)IVR-116, A/Panama/2007/99 (H₃N₂) Resvir-17, respectively and of 36 μg/mlfor B/Johannesburg/5/97. Tween 80 and Triton X-100 were adjusted to 580μg/ml and 90 μg/ml, respectively. The final volume was adjusted to 3 lwith phosphate buffered saline. The trivalent pool was filtered endingwith 0.8 μm cellulose acetate membrane to obtain the trivalent finalbulk. Trivalent final bulk was filled into syringes at least 0.5 mL ineach. TABLE 5 Comparison of time dependent HA content (μg/ml) measuredby SRD in trivalent final bulks. 0 4 weeks 6 months Vaccine formul.Strain months at 30° C. at 2-8° C. Influenza vaccine A/NCal/20/99 31 3230 without stabilizer A/Pan/2007/99 31 34 33 B/Joh/5/99* 35 25 31Influenza vaccine A/NCal/20/99 34 35 34 containing A/Pan/2007/99 33 3334 alpha-tocopherol B/Joh/5/99** 29 25 28 succinate

Example 7 Preparation of Influenza Virus Antigen Preparation UsingSodium Lauryl Sulfate as a Stabiliser for a Preservative-Free Vaccine(Thiomersal-Reduced Vaccine)

[0170] Monovalent Whole Virus Concentrate of B/Johannesburg/5/99 wasobtained as described in Example 1.

[0171] Sucrose Gradient Centrifugation with Sodium Deoxycholate

[0172] The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v). The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v)and is strain dependent. The flow rate is 8-15 litres/hour.

[0173] At the end of the centrifugation, the content of the rotor isrecovered by three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

[0174] Sterile Filtration

[0175] A sample of fraction 2 of 10 ml was taken for further processing.The split virus fraction is filtered on filter membranes ending with a0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween 80 and0.5 mM sodium lauryl sulfate is used for dilution. The final volume ofthe filtered fraction 2 is 5 times the original fraction volume.

[0176] Inactivation

[0177] The filtered monovalent material is incubated at 22±2° C. for atmost 84 hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffered saline containing 0.025% (w/v) Tween 80and 0.5 mM sodium laurylsulfate is then added in order to reduce thetotal protein content down to max. 250 μg/ml. Formaldehyde is added to afinal concentration of 50 μg/ml and the inactivation takes place at 20°C.±2° C. for at least 72 hours.

[0178] Ultrafiltration

[0179] The inactivated split virus material is concentrated at least 2fold in a ultrafiltration unit, equipped with cellulose acetatemembranes with 20 kDa MWCO. The Material is subsequently washed with 4volumes phosphate buffered saline containing 0.01% (w/v) Tween and 0.5mM sodium lauryl sulfate.

[0180] Final Sterile Filtration

[0181] The material after ultrafiltration is filtered on filtermembranes ending with a 0.2 μM membrane. Filter membranes are rinsed andthe material is diluted if necessary that the protein concentration doesnot exceed 500 μg/ml with phosphate buffered saline containing 0.01%(w/v) Tween 80 and 0.5 mM sodium lauryl sulfate.

[0182] Storage

[0183] The monovalent final bulk is stored at 2-8° C. TABLE 7 Comparisonof time dependent HA content measured by SRD in monovalent final bulks.After 4 weeks at stabiliser production 30° C. B/Johannesburg/5/99 None*182 139 (77%) B/Johannesburg/5/99 Sodium lauryl 288 264 (92%) sulfate

Example 8 Preparation of Influenza Virus Antigen Preparation UsingPlantacare or Laureth-9 as a Stabiliser for a Preservative-Free Vaccine(Thiomersal-Reduced Vaccine)

[0184] Monovalent Whole Virus Concentrate of B/Yamanashi/166/98 wasObtained as Described in Example 1.

[0185] Fragmentation

[0186] The monovalent whole influenza virus concentrate is diluted to aprotein concentration of 1,000 μg/ml with phosphate buffered saline pH7.4. Either Plantacare® 2000 UP or Laureth-9 is added to a finalconcentration of 1% (w/v). The material is slightly mixed for 30 min.Then the material is overlayed on a sucrose cushion 15% (w/w) in abucket. Ultracentrifugation in a Beckman swing out rotor SW 28 isperformed for 2 h at 25,000 rpm and 20° C.

[0187] Sterile Filtration

[0188] A supernatant was taken for further processing. The split virusfraction is filtered on filter membranes ending with a 0.2 μm membrane.

[0189] Inactivation

[0190] Phosphate buffered saline is added if necessary in order toreduce the total protein content down to max. 500 μg/ml. Formaldehyde isadded to a final concentration of 100 μg/ml and the inactivation takesplace at 20° C.±2° C. for at least 6 days.

[0191] Ultrafiltration

[0192] Tween 80 and Triton X 100 is adjusted in the inactivated materialto 0.15% and 0.02% respectively. The inactivated split virus material isconcentrated at least 2 fold in a ultrafiltration unit, equipped withcellulose acetate membranes with 30 kDa MWCO. The Material issubsequently washed with 4 volumes phosphate buffered saline.

[0193] Final Sterile Filtration

[0194] The material after ultrafiltration is filtered on filtermembranes ending with a 0.2 μm membrane. Filter membranes are rinsed andthe material is diluted that the protein concentration does not exceed500 μg/ml with phosphate buffered saline

[0195] Storage

[0196] The monovalent final bulk is stored at 2-8° C. TABLE 8 Comparisonof time dependent HA content measured by SRD in monovalent final bulks.After 4 weeks at stabiliser production 30° C. B/Yamanashi/166/98 None143 98 (68%)  B/Yamanashi/166/98 Plantacare ® 2000 476 477 (100%)  UPB/Yamanashi/166/98 Laureth-9 468 494 (>100%)

Example 9 Clinical Testing of α-Tocopherol Stabilised Influenza Vaccine(Reduced Thiomersal) in the Elderly Via ID and IM Administration

[0197] A Preparation of Influenza Virus Antigen Preparation

[0198] Monovalent split vaccine was prepared according to the followingprocedure.

[0199] Preparation of Virus Inoculum

[0200] On the day of inoculation of embryonated eggs a fresh inoculum isprepared by mixing the working seed lot with a phosphate buffered salinecontaining gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25μg/ml. (virus strain-dependent). The virus inoculum is kept at 2-8° C.

[0201] Inoculation of Embryonated Eggs

[0202] Nine to eleven day old embryonated eggs are used for virusreplication. Shells are decontaminated. The eggs are inoculated with 0.2ml of the virus inoculum. The inoculated eggs are incubated at theappropriate temperature (virus strain-dependent) for 48 to 96 hours. Atthe end of the incubation period, the embryos are killed by cooling andthe eggs are stored for 12-60 hours at 2-8° C.

[0203] Harvest

[0204] The allantoic fluid from the chilled embryonated eggs isharvested. Usually, 8 to 10 ml of crude allantoic fluid is collected peregg.

[0205] Concentration and Purification of Whole Virus From AllantoicFluid

[0206] 1. Clarification

[0207] The harvested allantoic fluid is clarified by moderate speedcentrifugation (range: 4000-14000 g).

[0208] 2. Adsorption step

[0209] To obtain a CaHPO₄ gel in the clarified virus pool, 0.5 mol/LNa₂HPO₄ and 0.5 mol/L CaCl₂ solutions are added to reach a finalconcentration of CaHPO₄ of 1.5 g to 3.5 g CaHPO₄/litre depending on thevirus strain.

[0210] After sedimentation for at last 8 hours, the supernatant isremoved and the sediment containing the influenza virus is resolubilisedby addition of a 0.26 mol/L EDTA-Na₂ solution, dependent on the amountof CaHPO₄ used.

[0211] 3. Filtration

[0212] The resuspended sediment is filtered on a 6 μm filter membrane.

[0213] 4. Sucrose Gradient Centrifugation

[0214] The influenza virus is concentrated by isopycnic centrifugationin a linear sucrose gradient (0.55% (w/v)) containing 100 μg/mlThiomersal. The flow rate is 8-15 litres/hour.

[0215] At the end of the centrifugation, the content of the rotor isrecovered by four different fractions (the sucrose is measured in arefractometer): fraction 1 55-52% sucrose fraction 2 approximately52-38% sucrose fraction 3 38-20% sucrose* fraction 4 20-0% sucrose

[0216] For further vaccine preparation, only fractions 2 and 3 are used.

[0217] Fraction 3 is washed by diafiltration with phosphate buffer inorder to reduce the sucrose content to approximately below 6%. Theinfluenza virus present in this diluted fraction is pelleted to removesoluble contaminants.

[0218] The pellet is resuspended and thoroughly mixed to obtain ahomogeneous suspension. Fraction 2 and the resuspended pellet offraction 3 are pooled and phosphate buffer is added to obtain a volumeof approximately 40 litres, a volume appropriate for 120,000 eggs/batch.This product is the monovalent whole virus concentrate.

[0219] 5. Sucrose Gradient Centrifugation with Sodium Deoxycholate

[0220] The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v) and Tocopherol succinate is added for B-strain-viruses up to 0.5mM. The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v) andis strain dependent. The flow rate is 8-15 litres/hour.

[0221] At the end of the centrifugation, the content of the rotor isrecovered by three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

[0222] 6. Sterile Filtration

[0223] The split virus fraction is filtered on filter membranes endingwith a 0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween80 and (for B strain viruses) 0.5 mM Tocopherol succinate is used fordilution. The final volume of the filtered fraction 2 is 5 times theoriginal fraction volume.

[0224] 7. Inactivation

[0225] The filtered monovalent material is incubated at 22±2° C. for atmost 84 hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffer containing 0.025% (w/v). Tween 80 is thenadded in order to reduce the total protein content down to max. 250μg/ml. For B strain viruses, a phosphate buffered saline containing0.025% (w/v) Tween 80 and 0.25 mM Tocopherol succinate is applied fordilution to reduce the total protein content down to 250 μg/ml.Formaldehyde is added to a final concentration of 50 μg/ml and theinactivation takes place at 20° C.±2° C. for at least 72 hours.

[0226] 8. Ultrafiltration

[0227] The inactivated split virus material is concentrated at least 2fold in a ultrafiltration unit, equipped with cellulose acetatemembranes with 20 kDa MWCO. The Material is subsequently washed withphosphate buffer containing 0.025% (w/v) Tween 80 and following withphosphate buffered saline containing 0.01% (w/v) Tween. For B strainvirus a phosphate buffered saline containing 0.01% (w/v) Tween 80 and0.1 mM Tocopherol succinate is used for washing.

[0228] 9. Final Sterile Filtration

[0229] The material after ultrafiltration is filtered on filtermembranes ending with a 0.2 μm membrane. Filter membranes are rinsed andthe material is diluted if necessary such that the protein concentrationdoes not exceed 1,000 μg/ml but haemagglutinin concentration exeeds 180μg/ml with phosphate buffered saline containing 0.01% (w/v) Tween 80 and(for B strain viruses) 0.1 mM Tocopherol succinate.

[0230] 10. Storage

[0231] The monovalent final bulk is stored at 2-8° C. for a maximum of18 months.

[0232] B Preparation of Influenza Vaccine

[0233] Monovalent final bulks of three strains, A/New Caldonia/20/99(H1N1) IVR-116, A/Panama/2007/99 (H₃N₂) Resvir-17 andB/Johannesburg/5/99 were produced according to the method described inpart A above.

[0234] Pooling

[0235] The appropriate amount of monovalent final bulks were pooled to afinal HA-concentration of 60 μg/ml for A/New Caldonia/20/99 (H1N1)IVR-116, A/Panama/2007/99 (H₃N₂) Resvir-17, respectively and of 68 μg/mlfor B/Johannesburg/5/99. Tween 80, Triton X-100 and Tocopherol succinatewere adjusted to 1,000 μg/ml, 110 μg/ml and 160 μg/ml, respectively. Thefinal volume was adjusted to 3 l with phosphate buffered saline. Thetrivalent pool was filtered ending with 0.8 μm cellulose acetatemembrane to obtain the trivalent final bulk. Trivalent final bulk wasfilled into syringes at least 0.165 mL in each.

[0236] Vaccine Administration

[0237] The vaccine was supplied in pre-filled syringes and wasadministered intradermally in the deltoid region. The intradermal (ID)needle was as described in EP1092444, having a skin penetration limiterto ensure proper intradermal injection. Since formation of a wheal(papule) at the injection site demonstrates the good quality of IDadministration, the investigator with the subject measured the exactsize of the wheal 30 minutes after vaccination.

[0238] One dose (100 μl) contained the following components:HEMAGGLUTININ FROM THREE INFLUENZA STRAINS A/NEW CALEDONIA/20/99 6.0 μgA/PANAMA/2007/99 6.0 μg B/JOHANNESBURG 5/99 6.0 μg THIOMERSALPRESERVATIVE 0.4 μg-0.8 μg

[0239] B The above vaccine was compared a standard trivalent splitinfluenza vaccine:

[0240] Fluarix™. The Fluarix vaccine was supplied in pre-filled syringesand was administered intramuscularly in the deltoid muscle. A needle ofat least 2.5 cm/1 inch in length (23 gauge) was used to ensure properintramuscular injection.

[0241] One dose (0.5 ml) contained the following components:HEMAGGLUTININ FROM THREE INFLUENZA STRAINS A/NEW CALEDONIA/20/99 15.0 μgA/PANAMA/2007/99 15.0 μg B/JOHANNESBURG 5/99 15.0 μg THIOMERSALPRESERVATIVE 50.0 μg

[0242] Results

[0243] The mean age of the total cohort at the time of vaccineadministration was 70.4±6.2 years Standard Deviation (S.D.), thefemale/male ratio was 1.7:1. Immunogenicity results: Analysis of derivedimmunogenicity variables was as follows: Flu-red ID Fluarix ™ Variable(N = 65) IM (N = 65) GMT GMT LL UL GMT LL UL A/NEW PRE 99.5 76.9 128.790.0 70.1 115.7 CALEDONIA POST 165.1 129.2 211.0 174.3 133.3 227.9A/PANAMA PRE 75.5 54.7 104.2 69.2 51.9 92.4 POST 128.6 99.1 166.8 164.3126.0 214.1 B/ PRE 236.0 187.7 296.8 222.6 176.9 280.2 JOHANNESBURG POST341.2 276.0 421.7 402.4 312.1 518.9 Seroconversion rate % LL UL % LL ULA/ 15.4 7.6 26.5 18.5 9.9 30.0 NEW CALEDONIA A/PANAMA 20.0 11.1 31.829.2 18.6 41.8 B/ 9.2 3.5 19.0 16.9 8.8 28.3 JOHANNESBURG Conversionfactor GMR LL UL GMR LL UL A/NEW 1.7 1.4 2.0 1.9 1.6 2.3 CALEDONIAA/PANAMA 1.7 1.4 2.1 2.4 1.9 3.0 B/ 1.4 1.2 1.7 1.8 1.5 2.1 JOHANNESBURGSeroprotection rate % LL UL % LL UL A/NEW PRE 87.7 77.2 94.5 90.8 81.096.5 CALEDONIA POST 92.3 83.0 97.5 96.9 89.3 99.6 A/PANAMA PRE 75.4 63.185.2 81.5 70.0 90.1 POST 90.8 81.0 96.5 93.8 85.0 98.3 B/ PRE 98.5 91.7100.0 96.9 89.3 99.6 JOHANNESBURG POST 100.0 94.5 100.0 98.5 91.7 100.0

[0244] Injection site pain, reported by 10/65 (15.4%) vaccinees, was themost common symptom following IM administration of Fluarixm. In the IDgroup, pain was reported by 3/65 (4.6%) vaccinees. This difference wasstatistically significant (p=0.038; Fisher exact test). Accordingly theID delivery of a thiomersal reduced product is preferred.

[0245] Conclusions

[0246] Both ID and IM administration of a thio-reduced flu vaccine in anelderly population can provide 100% seroprotection.

[0247] A comparable response to vaccination in terms of geometric meantiters, seroprotection rates, seroconversion rates and conversionfactors was found in IM and ID vaccinated individuals where the ID groupreceived 2.5-fold less antigen.

[0248] There was no discernible difference in the overall incidence ofvaccine-related solicited/unsolicited systemic symptoms in the twotreatment groups.

Example 10 Intradermal Delivery of a Thiomersal-Reduced InfluenzaVaccine

[0249] Immunogenicity of the thiomersal reduced split influenza vaccineprepared as described in Example 9 (except that the pooling was doneindependently and the vaccine was not filled into syringes) was assessedby ID delivery in guinea pigs using a standard needle.

[0250] Groups of 5 animals each were primed intranasally with wholeinactivated trivalent influenza virus containing 5 μg of each HA in atotal volume of 200 μl. Twenty-eight days after priming the animals werevaccinated by either the intradermal or intramuscular routes.Intradermal doses containing 0.1, 0.3, or 1.0 μg trivalentthiomersal-reduced split Flu in 0.1 ml were administered in the back ofthe guinea pig using a standard needle An intramuscular dose of 1.0 μgtrivalent thiomersal-reduced split Flu was administered in the hind legof the guinea pig in a volume of 0.1 ml. The groups were as follows:

[0251] Group 1—0.1 μg trivalent thiomersal-reduced split Flu ID;

[0252] Group 2—0.3 μg trivalent thiomersal-reduced split Flu ID;

[0253] Group 3—1.0 μg trivalent thiomersal-reduced split Flu ID

[0254] Group 4—1.0 μg trivalent thiomersal-reduced split Flu IM

[0255] Fourteen days after vaccination the animals were bled and theantibody titers induced by the vaccination were assessed using astandard hemagglutination inhibition assay (HI). The results are shownin FIG. 1. Strong HI responses to all three strains were induced byvaccination. No clear dose response was noted suggesting that very lowdoses of thiomersal-reduced antigen can still induce very potent HIantibody responses when administered by the ID route. There was nosignificant difference between the HI titers induced by ID or IMvaccination. Thus, the results obtained in guinea pigs confirmed thatthe thimerosal-reduced trivalent split influenza antigens induce similarlevels of HI antibodies in animals when delivered by the ID routecompared to the IM route.

Example 11 Intradermal Delivery of a Thiomersal-Reduced, AdjuvantedInfluenza Vaccine

[0256] Protocol

[0257] Guinea pigs were primed on Day 0 with 5 μg trivalent wholeinactivated Flu virus in 200 μl, intranasally.

[0258] Vaccination—Day 28—Vaccine containing 0.1 μg HA per straintrivalent split Flu prepared as described in Example 9 (except that thepooling step resulted in a final concentration for each antigen of 1.0μg/ml to give a dose of 0.1 μg in 100 μl compared to 60 μg/ml in Example9). The final trivalent formulation was administered intradermally usingtuberculin syringes, either adjuvanted or unadjuvanted, in 100 μl.

[0259] Bleeding—Day 42.

[0260] The effect of adjuvantation was assessed by measuring antibodyresponses by HI assay (day 0, 28, 42).

[0261] All ID experiments were carried out using a standard needle.

[0262] Results

[0263] G1-G5 refer to 5 groups of guinea pigs, 5 per group. G1 Splittrivalent thiomersal reduced 0.1 μg G2 Split trivalent thio red 0.1 μg +3D-MPL 50 μg G3 Split trivalent thio red 0.1 μg + 3D-MPL 10 μg G4 Splittrivalent thio red 0.1 μg + 3D-MPLin 50 μg + QS21 50 μg G5 Splittrivalent thio red 0.1 μg + 3D-MPLin 10 μg + QS21 10 μg

[0264] Note 3D-MPLin+QS21 refers to an adjuvant formulation whichcomprises a unilamellar vesicle comprising cholesterol, having a lipidbilayer comprising dioleoyl phosphatidyl choline, wherein the QS21 andthe 3D-MPL are associated with, or embedded within, the lipid bilayer.Such adjuvant formulations are described in EP 0 822 831 B, thedisclosure of which is incorporated herein by reference. HI Titresanti-A/New Caledonia/20/99 NC Pre-immun Pre-boost Post-boost G1 5 10 92G2 5 10 70 G3 5 11 121 G4 7 9 368 G5 5 10 243

[0265] HI Titres anti-A/Panama/2007/99 P Pre-immun Pre-boost Post-boostG1 5 485 7760 G2 5 279 7760 G3 5 485 8914 G4 7 485 47051 G5 5 320 17829

[0266] HI Titres anti-B/Johannesburg/5/99 J Pre-immun Pre-boostPost-boost G1 5 23 184 G2 5 11 121 G3 5 11 70 G4 6 15 557 G5 5 13 320

[0267] Thus, whether adjuvanted or unadjuvanted the thiomersal-reducedtrivalent split Flu antigen is a potent immunogen and capable ofinducing strong HI responses when administered by the ID or IM route.These responses appear to be at least as potent as the responses inducedby the standard Fluarix preparation.

Example 12 Comparison of Thiomersal-Containing and Thiomersal-FreeVaccine Delivered Intradermally in Pigs

[0268] In order to assess the immunogenicity of the split Flu vaccine(plus and minus thiomersal) administered by the ID route the primed pigmodel was used. As the vast majority of the population has experiencedat least one infection with influenza an influenza vaccine must be ableto boost a pre-existing immune response. Therefore animals are primed inan effort to best simulate the human situation.

[0269] In this experiment 4 week old pigs were primed by the intranasalroute. Six groups of five animals each were primed as follows:

[0270] Group 1—two primings of trivalent whole inactivated virus (50 μgeach HA) at day 0 and 14; Group 2—two primings of trivalent wholeinactivated virus (50 μg each HA) at day 0 and 14; Group 3—singlepriming with trivalent whole inactivated virus (50 μg each HA) at day 0;Group 4—two primings of trivalent whole inactivated virus (25 μg eachHA) at day 0 and 14; Group 5—single priming of trivalent wholeinactivated virus (25 μg each HA) at day 0; Group 6—two primings oftrivalent whole inactivated virus (12.5 μg each HA) at day 0 and 14.

[0271] On day 28 post final priming, the animals were vaccinated with 3μg each HA trivalent split antigen (strains A/New Calcdonia H1N1,A/Panama H₃N₂, and B/Johannesburg) in 100 μl by the ID route. Group 1received standard Fluarix™ containing thiomersal preservative as vaccineantigen. All other groups received the preservative-free antigen.

[0272] The HI results obtained in this experiment are shown in FIG. 2(Anti-Influenza Hemagglutination Inhibition Titers Induced in PigsPrimed with a Variety of Antigen Doses and Vaccinated with 3 MicrogramsTrivalent Influenza Antigen Plus or Minus Thiomersal by the IntradermalRoute).

[0273] Relatively low HI titers are induced to the B strain in thisexperiment and the background against the A/H₃N₂ strain is high. Abeneficial effect in terms of response to vaccination is observed whenthe priming dose is reduced. In almost all cases, reduction in theantigen concentration or number of priming doses (from the two primingswith 50 μg) resulted in a heightened response to vaccination. While theresponse of the animals in Groups 1 and 2, which were primed twice with50 μg, to vaccination is not so evident, it appears that thepreservative-free antigen (Group 2) functions at least as well asFluarix™ (Group 1) under these conditions. A strong response tovaccination with preservative-free trivalent influenza antigenadministered by the ID route in the alternatively primed animals (Groups3-6) is clear and this response is seen even in the B strain, althoughthe HI titers remain low.

1. An inactivated influenza virus preparation comprising ahaemagglutinin antigen stabilised in the absence of thiomersal, or atlow levels of thiomersal, wherein the haemagglutinin is detectable by aSRD assay, and wherein the preparation comprises α-tocopherol succinatein a sufficient amount to stabilise the HA.
 2. The inactivated influenzavirus preparation according to claim 1 wherein the α-tocopherolsuccinate is present at a concentration between 1 ug/ml and 10 mg/ml. 3.The inactivated influenza virus preparation according to claim 2 whereinthe α-tocopherol succinate is present at a concentration between 10 and500 ug/ml.
 4. The inactivated influenza virus preparation according toclaim 1 wherein the influenza virus antigen preparation is selected fromthe group consisting of split virus antigen preparations, subunitantigens, chemically or otherwise inactivated whole virus.
 5. Theinactivated influenza virus preparation according to claim 4 wherein theinfluenza antigen preparation is a split virus antigen preparation. 6.The inactivated influenza virus preparation according to claim 1comprising both A and B strain haemagglutinin.
 7. The inactivatedinfluenza virus preparation according to claim 6 which is a trivalentinfluenza virus preparation.
 8. The inactivated influenza viruspreparation according to claim 1 comprising stabilised B straininfluenza HA.
 9. An influenza vaccine comprising the influenza viruspreparation according to any one of claim
 1. 10. The influenza vaccineaccording to claim 9 wherein the concentration of haemagglutinin antigenfor the or each strain of influenza is 1-1000 μg per ml, as measured bya SRD assay.
 11. An influenza vaccine according to claim 9, additionallycomprising an adjuvant.
 12. A method for preparing a stablehaemagglutinin antigen which method comprises purifying the antigen inthe presence of α-tocopherol or a derivative thereof such asα-tocopherol succinate.
 13. A method for prophylaxis of influenzainfection or disease in a subject which method comprises administeringto the subject a vaccine according to claim
 9. 14. A method according toclaim 13, in which vaccine delivery is intradermal, intranasal,intramuscular, oral or subcutaneous. 15-35. cancelled.